Neddylation orchestrates the complex transcriptional and posttranscriptional program that drives Schwann cell myelination

Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the posttranslational modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a notable absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Our data indicate that neddylation exerts a global influence on the complex transcriptional and posttranscriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signaling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.

(J) Immunoblot analyses show that pharmacological inhibition of neddylation using MLN4924 does not reduce db cAMP-mediated upregulation of MPZ in primary rat Schwann cell cultures.Primary rat Schwann cells were cultured under myelinogenic conditions (db cAMP treatment) for 48h leading to an upregulation of MPZ protein levels.MLN4924 treatment for a further 48h was not sufficient to reduce MPZ levels.
Efficacy of neddylation inhibition by MLN4924 is shown by reduced levels of neddylated proteins.

Fig. S2 :
Fig. S2: Nae1 deletion in vivo blocks myelin sheath formation (A -C) Schematic diagram depicting the strategy for generating Schwann cell-specific Nae1 conditional mice (Nae1 cKO).(A) Nae1 floxed mice were crossed with Schwann cell-restricted MPZ-Cre transgenic mice to remove the critical exon 4 of Nae1, resulting in a frameshift and a premature stop codon in exon 5. Primers for demonstrating recombination efficiency in (B) and (C) are indicated as arrows in the construct.LoxP sites are denoted by green triangles.(B) Genomic DNA was extracted from sciatic nerves and analyzed by PCR to detect a recombined band at ~320 bp in Nae1 cKO mice and an unrecombined band at ~1000 bp in control mice (P1 and P2).(C) RT-qPCR analysis of Nae1 mRNA levels using a pair of primers (P3 and P4) recognizing exon 4 of Nae1 gene.Data are presented as mean ± SEM; n = 4 WT and 6 Nae1 cKO mice.Two-tailed unpaired Student's t-test (t = 9.591, d.f.= 8).(D) Graph shows quantification of nerve area in control and Nae1 cKO mice at indicated ages.Data are presented as mean ± SEM; n= 3-6; Two-way ANOVA with Sidak's multiple-comparisons test.(E) Densitometric quantification of protein levels from Fig. 3E showing reduced levels of myelin proteins MPZ, CNP and MBP in Nae1 cKO mice.Data are presented as mean ± SEM; n = 5-7.One-way ANOVA with Tukey's multiple-comparisons test [MPZ: F(2, 15) = 20.01;CNP: F(2, 12) = 17.18;MBP: F(2, 16) = 18.54].(F) Representative EM pictures showing Remak bundles in control and Nae1 cKO P28 sciatic nerves.Numerous large Remak bundles (asterisk) are seen in Nae1 cKO nerves, as well as large diameter axons (> 1 µm) that are still present within these families (arrowheads).(G) Graph shows quantification of number of axons per Remak bundle in control and Nae1 cKO mice at P7 and P28.Data are presented as mean ± SEM; n= 3-6; Two-way ANOVA with Sidak's multiplecomparisons test.(H) Graph shows the percentage of Remak bundles that contain 1 or more axon of large diameter (> 1 µm) at P7 and P28.Data are presented as mean ± SEM; n= 3-6; Two-way ANOVA with Sidak's multiplecomparisons test.(I) Representative EM pictures showing a demyelinating Schwann cell in Nae1 cKO sciatic nerves.(M= myelin ovoids; A = axon, and arrowhead indicate the Schwann cell basal lamina).Graph shows quantification of demyelinating Schwann cell profiles per nerve in control and Nae1 cKO mice at indicated ages.Data are presented as mean ± SEM; n= 3-6; Two-way ANOVA with Sidak's multiple-comparisons test.(J) Representative EM pictures showing an abnormal Schwann cell profile (e.g.Schwann cell myelinating two axons) in Nae1 cKO sciatic nerves.Graph shows quantification of abnormal Schwann cell myelin profiles per nerve in control and Nae1 cKO mice at indicated ages.Data are presented as mean ± SEM; n= 3-6; Two-way ANOVA with Sidak's multiple-comparisons test.

Fig. S4 :
Fig. S4: KEGG pathway diagram that represents the relationships of gene products in 'Ubiquitin mediated proteolysis' (A, B) KEGG pathway diagram that represents the relationships of gene products in 'Ubiquitin mediated proteolysis' -mmu04120 pathway (https://www.genome.jp/entry/mmu04120). (A) Protein ubiquitination plays an important role in eukaryotic cellular processes.It mainly functions as a signal for 26S proteasome dependent protein degradation.The addition of ubiquitin to proteins being degraded is performed by a reaction cascade consisting of three enzymes, named E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase).(B) Each E3 has specificity to its substrate, or proteins to be targeted by ubiquitination.E3s are classified into four types: HECT type, U-box type, single RINGfinger type, and multi-subunit RING-finger type.Multi-subunit RING-finger E3s are exemplified by cullin-Rbx E3s (CRLs) and APC/C.They consist of a RING-finger-containing subunit (RBX1 or RBX2) that functions to bind E2s, a scaffold-like cullin molecule, adaptor proteins, and a target recognizing subunit that binds substrates.Genes marked in red correspond to the significantly differentially expressed proteins between WT and Nae1 cKO mice of E1 and E2 enzymes, and different types of E3 ubiquitin ligases.CRLs are the most represented categories in the significantly differentially expressed proteins.

Fig. S5 :
Fig. S5: Neddylation regulates expression of c-Jun and Sox2 in Schwann cells.(A) Densitometric quantification of protein levels from Fig. 6D showing upregulation of c-Jun and Sox2 in Nae1 cKO mice.Data are presented as mean ± SEM; n=5-6; one-way ANOVA with Tukey's multiplecomparisons test [c-Jun: F(2, 15) = 6.702;Sox2: F(2, 12) = 8.481].(B, C) Immunoblot analyses showing (B) c-Jun and (C) Sox2 protein levels in total sciatic nerve lysates from NB and P7 nerves from control and Nae1 cKO mice.Densitometric quantification of c-Jun and Sox2 protein levels are shown at indicated ages.Data are presented as mean ± SEM; n=3-4; Two-tailed unpaired Student's t-test.(D, E) RT-qPCR showing expression of (D) c-Jun and (E) Sox2 mRNA levels in primary Schwann cells pretreated with db cAMP for 24h, which strongly reduces c-Jun and Sox2 mRNA levels, followed by treatment with vehicle and MLN4924.Vehicle (DMSO) or MLN4924 treatment had no effect on c-Jun and Sox2 mRNA levels.Red dot represents levels before db cAMP treatment and Time 0 refers to levels 24h after db cAMP supplementation).(F, G) Immunoblot analyses of (F) c-Jun and (G) Sox2 protein levels in primary Schwann cells pretreated with db cAMP for 24h, which strongly reduces c-Jun and Sox2 protein levels, followed by treatment with vehicle and MLN4924.Vehicle treatment (DMSO) had no effect on c-Jun and Sox2 protein levels, whereas MLN4924 treatment led to a remarkable upregulation of c-Jun and Sox2 protein.Time 0 refers to control cultures before db cAMP treatment.Graphs show densitometric quantification of c-Jun and Sox2 protein levels in vehicle or MLN4924-treated cultures.Data are presented as mean ± SEM; n=3.Two-way ANOVA with Sidak's multiple comparison test.Dotted lines represent non-linear regression analysis (one-phase decay) of c-Jun and Sox2 degradation.*p<0.05;**p<0.01;****p<0.0001b-actin is used as loading control for immunoblots (B, F, G).

Fig. S6 :
Fig. S6: Rescue experiments show that neddylation regulates parallel pathways in Schwann cells (A) Immunoblot analyses showing that treatment with rapamycin does not prevent the MLN4924-induced upregulation of c-Jun and Sox2, downregulation of EGR2, and inhibition of the Hippo-Yap signalling pathway in primary rat Schwann cells cultured under myelinogenic conditions (db cAMP treatment).bactin is used as loading control for immunoblots.(B) Immunoblot analyses showing that treatment with XMU-MP-1 does not prevent the MLN4924-induced upregulation of c-Jun and Sox2, downregulation of EGR2, and hyperactivation of the mTOR pathway in primary rat Schwann cells cultured under myelinogenic conditions (db cAMP treatment).b-actin is used as loading control for immunoblots.(C) Immunoblot analyses showing that silencing of c-Jun does not prevent the MLN4924-induced upregulation of Sox2, downregulation of EGR2, hyperactivation of the mTOR pathway, and inhibition of the Hippo-Yap signalling pathway in primary rat Schwann cells cultured under myelinogenic conditions (db cAMP treatment).b-actin is used as loading control for immunoblots.(D) Immunoblot analyses showing that silencing of Sox2 does not prevent the MLN4924-induced upregulation of c-Jun, downregulation of EGR2, hyperactivation of the mTOR pathway, and inhibition of the Hippo-Yap signalling pathway in primary rat Schwann cells cultured under myelinogenic conditions (db cAMP treatment).b-actin is used as loading control for immunoblots.(E) Immunoblot analyses showing that treatment with MG132 does not prevent the MLN4924-induced upregulation of c-Jun and Sox2, hyperactivation of the mTOR pathway, and inhibition of the Hippo-Yap signalling pathway in primary rat Schwann cells cultured under myelinogenic conditions (db cAMP treatment).b-actin is used as loading control for immunoblots.(F) Graph shows densitometric quantification from Fig. 9G.Data are presented as mean ± SEM; n = 4-6.